For cancers to grow and thrive, normal cells must undergo changes in cell cycle regulation that allow them to proliferate rapidly and indefinitely. An incomplete understanding of these cell cycle adaptations hinders the development of cancer therapies. The Rb family proteins (Rb, p107, and p130), which regulate cell cycle arrest in normal cells by forming repressive complexes with E2F transcription factors, are rendered inactive in most, if not all, human cancers. Our long-term goal is to understand the molecular events that result in tumorigenesis upon loss of Rb family function. Among the nine E2F transcription factors, E2F4 is the major repressor E2F in adult tissues and organs, where it represses cell cycle genes in association with Rb family members. Here we hypothesize that in contexts in which Rb family proteins are inactive, E2F4 can act as a transcriptional activator driving rapid cell cycle progression. This hypothesis is supported by emerging evidence that E2F4 is highly expressed in multiple cancer types, and is nuclear and bound to some target genes in rapidly proliferating cells. However, we lack a complete understanding of which genes and pathways are regulated by E2F4 as an activator, and it is equally puzzling how E2F4, which lacks a nuclear localization signal, can enter the nucleus and bind to its targets in the absence of functional Rb family members. To investigate non-canonical roles for E2F4, we propose to use mouse embryonic stem cells (ESCs) as a cellular model. ESCs provide a genetically stable and tractable system with remarkable similarities to cancer cells, including functional inactivatio of the Rb family and hyperactivation of genes involved in metabolism and cell cycle progression. In addition, our preliminary studies indicate that E2F4 binds to highly expressed genes in ESCs in an Rb family-independent manner. We also found that loss of E2F4 leads to the downregulation of cell cycle targets. Based on these observations, we first propose to identify all the targets directly activated by E2F4 in ESCs using unbiased genomic approaches (ChIP-Seq and RNA-Seq). Second, we will identify the co- factors that regulate the nuclear localization and transcriptional activity of E2F4 in the absence of the Rb family proteins, using a combination of bioinformatics approaches and molecular assays. Third, we will investigate the role of E2F4 in the long-term self-renewal of ESCs by performing various cellular assays in gain- and loss-of-function experiments. This study will characterize a novel role for a canonical cell cycle repressor and will identify for the first time a functional role for an E2F family member in ESCs. Equally important, we expect that the results of the proposed experiments will directly inform research into the biology of cancer cells and other contexts where E2F4 appears to serve as a transcriptional activator.